Recombinant adeno-associated viruses (rAAV) are promising vectors for in vivo gene delivery. A number of naturally occurring serotypes and subtypes have been isolated from human and non-human primate tissues (Gao G et al., J Virol 78, 6381-6388 (2004) and Gao G et al., Proc Natl Acad Sci USA 99, 11854-11859 (2002), both of which are incorporated by reference herein). Among the newly-identified adeno-associated virus (AAV) isolates, AAV serotype 8 (AAV8) and AAV serotype 9 (AAV9) have gained much attention because rAAV vectors derived from these two serotypes can transduce various organs including the liver, heart, skeletal muscles and central nervous system with high efficiency following systemic administration via the periphery (Foust K D et al., Nat Biotechnol 27, 59-65 (2009); Gao et al, 2004, supra; Ghosh A et al., Mol Ther 15, 750-755 (2007); Inagaki K et al, Mol Ther 14, 45-53 (2006); Nakai H et al., J Virol 79, 214-224 (2005); Pacak C A et al., Circ Res 99, e3-e9 (2006); Wang Z et al., Nat Biotechnol 23, 321-328 (2005); and Zhu T et al., Circulation 112, 2650-2659 (2005), all of which are incorporated by reference herein).
The robust transduction by rAAV8 and rAAV9 vectors has been presumed to be ascribed to strong tropism for these cell types, efficient cellular uptake of vectors, and/or rapid uncoating of virion shells in cells (Thomas C E et al., J Virol 78, 3110-3122 (2004), incorporated by reference herein). In addition, emergence of capsid-engineered rAAV with better performance has significantly broadened the utility of rAAV as a vector toolkit (Asokan A et al., Mol Ther, published in advance of press doi:10.1038/mt.2011.287 (2012), incorporated by reference herein). Proof-of-concept for rAAV-mediated gene therapy has been shown in many preclinical animal models of human diseases. Phase I/II clinical studies have been initiated or completed for genetic diseases including hemophilia B (Manno C S et al., Nat Med 12, 342-347 (2006) and Nathwani A C et al., N Engl J Med 365, 2357-2365 (2011), both of which are incorporated by reference herein); muscular dystrophy (Mendell J R et al., N Engl J Med 363, 1429-1437 (2011), incorporated by reference herein); cardiac failure (Jessup M et al., Circulation 124, 304-313 (2011), incorporated by reference herein); blinding retinopathy (Maguire A M et al., Lancet 374, 1597-1605 (2009), incorporated by reference herein); and α1 anti-trypsin deficiency (Flotte T R et al., Hum Gene Ther 22, 1239-1247 (2011), incorporated by reference herein), among others.
Although rAAV vectors have widely been used in preclinical animal studies and have been tested in clinical safety studies, the current rAAV-mediated gene delivery systems remain suboptimal for broader clinical applications. The sequence of an AAV viral capsid protein defines numerous features of a particular AAV vector. For example, the capsid protein affects capsid structure and assembly, interactions with AAV nonstructural proteins such as Rep and AAP proteins, interactions with host body fluids and extracellular matrix, clearance of the virus from the blood, vascular permeability, antigenicity, reactivity to neutralizing antibodies, tissue/organ/cell type tropism, efficiency of cell attachment and internalization, intracellular trafficking routes, virion uncoating rates, among others. Furthermore, the relationship between a given AAV capsid amino acid sequence and the characteristics of the rAAV vector are unpredictable. Therefore, new rAAV vectors with altered capsid proteins are needed in order to maximize the benefit of AAV based therapies.